Date Published: March 14, 2019
Publisher: Public Library of Science
Author(s): Kerrie L. Ford, Maryam Anwar, Rachael Heys, Eltayeb Mohamed Ahmed, Massimo Caputo, Laurence Game, Barnaby C. Reeves, Prakash P. Punjabi, Gianni D. Angelini, Enrico Petretto, Costanza Emanueli, Carlo Gaetano.
This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.
Despite improvements in coronary revascularisation techniques, ischaemic heart disease (IHD) remains a leading cause of morbidity and mortality in more economically developed countries, and patients are at risk of developing post-intervention complications. Type 2 diabetes mellitus (T2DM), which is projected to affect 592 million people globally by 2035, is a major risk factor both for IHD and for severe acute post-surgical complications following coronary intervention, such as acute kidney injury. New therapeutic targets and better non-invasive biomarkers are needed to improve IHD treatment and the post-revascularization outcomes, particularly in the context of comorbidities such as type 2 diabetes.
Our results indicate that for technically demanding and costly applications such as RNA sequencing or other high-throughput screening methods, it is critical to validate the RNA extraction method for each sample type, as no single method is optimal for all. To the best of our knowledge, few similar systematic analyses have been performed on other cardiovascular clinical tissue sample types. Ip et al compared an organic extraction method with a solid-phase extraction method on archived right atrial appendage samples, however these samples are different in size and composition from the more clinically-relevant left ventricular biopsies obtained in the ARCADIA study.